Note: Descriptions are shown in the official language in which they were submitted.
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Culture medium for cultivating Hathewaya histolytica (or Clostridium
histolyticum) and the
production of one or more proteases
Introduction
The present invention relates to a liquid culture medium comprising yeast
extract, glycine,
arginine, glutamine, serine, threonine, magnesium ions, calcium ions, selenium
(e.g., in the
form of sodium selenite) and water. It further relates to a liquid feed
composition for use as
feed in a fed-batch process comprising at least yeast extract, threonine,
serine and water. It
relates to a kit of parts comprising the liquid culture medium and the liquid
feed composition,
the use of the liquid culture medium and the liquid feed composition for
cultivating Hathewaya
histolytica (previously Clostridium histolyticum) and obtaining one or more
proteases and
methods concerning the cultivation.
Discussion of prior art
Collagen is the major structural constituent of mammalian organisms and makes
up a large
portion of the total protein content of skin and other parts of the animal
body. In humans, it is
particularly important in the wound healing process and in the process of
natural aging.
Various skin traumas such as bumps, surgery, infection and accident are often
characterized
by the erratic accumulation of fibrous tissue that is rich in collagen and
having increased
proteoglycan content. In addition to the replacement of the normal tissue
which has been
damaged or destroyed, excessive and disfiguring deposits of new tissue
sometimes form during
the healing process.
Numerous diseases and conditions are associated with excess collagen
deposition and the
erratic accumulation of fibrous tissue rich in collagen. Such diseases and
conditions are
collectively referred to herein as ''collagen-mediated diseases". Collagenase
is an enzyme that
has the specific ability to digest collagen. Collagenase has been used to
treat a variety of
collagen-mediated diseases such as Peyronie's disease and Dupuytren's disease,
and to
remove necrotic tissue from wounds.
Furthermore, collagenase and other proteases (such as e.g. neutral protease
and/or
clostripain) are used in vitro for tissue dissociation and isolation of a
variety of cells, such as
e.g. pancreatic islet cells, hepatocytes and tumor cells. These cells find
multiple applications in
research and clinics. One common source of crude collagenase or mixtures of
proteases is from
a bacterial fermentation process, especially from the fermentation of
Hathewaya histolytica
(previously Clostridium histolyticum).
Hathewaya (Clostridium) is a genus of gram-positive bacteria. Hathewaya
(Clostridium)
bacteria are anaerobic and are common in the soil, water and in the intestinal
tracts of humans
and other animals. The species Hathewaya histolytica (Clostridium
histolyticum) is capable of
producing collagen degrading enzymes and other enzymes with proteolytic
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activity, such as e.g. collagenases (EC 3.4.24.3), neutral protease and
clostripain (EC 3.4.22.8).
Important products of the cultivation process are the bacterial collagenases
which possess
proteolytic activity against collagen. These enzymes have been classified by
Bond et al (Bond,
M.D., van Wart, H.E.; Biochemistry, 23, 3077-91 (1984)) as type I and II
collagenases (hereafter
"collagenase l" and "collagenase II") according to their relative activity.
The collagenases and
the other proteases are secreted into the culture medium and can be obtained
from the culture
supernatant.
In the biotech industry a number of enzymes for use in pharmaceutical
applications are
produced in large-scale fermentation processes. For example, Hathewaya
histolytica
(Clostridium histolyticum) is cultivated to produce proteases, including e.g.
collagenases,
neutral protease and/or clostripain. Usually, in such processes the culture
media (nutrient
media, fermentation media) comprise animal-derived components such as meat
(tissue)
peptones of either bovine or porcine origin. Because adventitious agents (e.g.
viruses)
originating from animal-derived growth media might be present in the final
product, it is
desirable to develop culture media free of mammalian pathogenic agents. There
is a particular
safety-related concern regarding prions and BSE. In recent years, regulatory
authorities
request these concerns to be addressed. Therefore, there is a tendency in the
industry towards
animal-free culture media.
In EP 2 133 415 Al a growth medium for Hathewaya histolytica (Clostridium
histolyticum) is
disclosed that comprises water, fish gelatin and a peptone from a non-
mammalian source,
whereby fish peptone is excluded. The only examples for a peptone from a non-
mammalian
source are plant peptones. This growth medium suffers from the fact that fish
gelatin and a
peptone from a non-mammalian source are complex ingredients that comprise a
large variety
of compounds, most of which are not known. Moreover, they are prepared from
natural
sources and are therefore subject to natural fluctuations in their contents.
Yeast extracts and plant peptones have recently been considered as substitutes
for animal-
derived culture media components. However, yeast extracts alone may not
provide all
nutrients necessary for sufficient growth of certain bacteria. EP 2 865 748 Al
discloses animal
product-free culture media for bacteria of the genus Clostridium (now
Hathewaya), especially
Clostridium histolyticum (now Hathewaya histolytica) comprising water, non-
animal origin
peptone, or its derivatives, yeast extract and the amino acids cysteine and
arginine. The non-
animal origin peptones are derived from plants (plant peptones). Vegetable
peptones are used
according to WO 2007/089851 Al for the fermentation of Clostridium
histolyticum (now
Hathewaya histolytica) to produce collagenase I and collagenase II. Plant
peptones have the
disadvantages that they are complex and undefined mixtures and that the
production of the
raw materials is often subject to large batch variability, especially if
weather conditions or
planting regions change. Moreover, the composition of plant peptones may
significantly vary
depending on the production process and the supplier. Thus, it is difficult or
just not possible
to obtain a constant product quality or to find alternative suppliers. For
these reasons, plant
peptones are not desired as ingredient for most industrial processes for the
cultivation of
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bacteria. This is especially true for the cultivation of Hathewaya histolytica
(Clostridium
histolyticum) and the production of proteases. Hathewaya histolytica
(Clostridium
histolyticum) secretes collagenase I, collagenase II, clostripain and neutral
protease during
cultivation. The activity of all these enzymes in the supernatant is strongly
influenced by the
compositions of the plant peptones used for cultivation and may vary by a
factor 5 or more,
depending on the source or raw material of a plant peptone, the manufacturer,
the production
process, and on the batch. As outlined above, such product variability and
dependency on a
single supplier are suboptimal for an industrial production process,
especially in the
pharmaceutical industry.
Purpose of the invention
The aim of this invention was to provide a liquid medium and/or a liquid feed
composition
which (i) supports growth of the bacteria and (ii) stimulates secretion of
enzymes with
proteolytic activity into the culture medium and (iii) reduces product and
process variability. It
is particularly preferred that the medium stimulates a high volume activity
(enzyme activity per
culture volume) in the culture supernatant. An additional purpose of the
present invention is
to provide a new type of culture medium that allows the cultivation of
bacteria without
addition of animal-derived components and/or plant derived components. The
medium should
furthermore have as few components as possible, in order to avoid any
contamination with
undesirable substances and to avoid overly complex influences on the
fermentation process.
It is especially desired that such culture medium allows for the effective
cultivation of the
species Hathewaya histolytica (Clostridium histolyticum) and for the
production of one or more
proteases, e.g. collagenase I, collagenase II, neutral protease and/or
clostripain. It is a further
object of the present invention to provide a supernatant of a Hathewaya
histolytica
(Clostridium histolyticum) liquid culture, which may be used forthe isolation
of the one or more
proteases comprised therein. It is also an object of the present invention to
provide a process
for the production of proteases wherein the degradation of the proteases is
low. In addition,
the culture medium may provide the following benefits: a) higher batch
reliability than culture
media comprising animal peptones or plant peptones; b) all components can be
obtained in
suitable and constant quality from multiple suppliers; c) the activity of
collagenase in the
supernatant of Hathewaya histolytica (Clostridium histolyticum) is 400 U / L
or more (PZ
activity).
Detailed description
In a first aspect, the present invention concerns a liquid culture medium
comprising, essentially
consisting of, or consisting of
2.5 to 100 g/L of yeast extract,
1.0 to 30 g/L of glycine,
1.0 to 35 g/L of argi nine,
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0.20 to 0.80 g/L of glutamine,
0.20 to 1.0 g/L of serine,
0.3 to 3.0 g/L of threonine,
0.4 to 12 mmol/L magnesium ions (Mg2), e.g. 100 to 3000 mg/L of magnesium
sulfate
he ptahydrate (Mg504 x 7 H20),
0.34 to 3.4 mmol/L calcium ions (Ca2+), e.g. 50 to 500 mg/L of calciumchloride
dihydrate
(CaCl2 x 2 H20).,
0.00095 to 0.0152 mmol/L selenium, e.g. 0.25 to 4.00 mg/L sodium selenite
pentahydrate
(Na2Se03 x 5 H20), and
water.
The term "essentially consists of" as used herein shall mean that the
composition (a)
necessarily includes the listed ingredients and (b) is open to unlisted
ingredients that do not
materially affect the basic and novel properties of the composition.
The liquid culture medium according to the present invention is suitable as
culture medium for
bacteria of the genus Hathewaya (Clostridium) and especially as culture medium
for
Hathewaya histolytica (Clostridium histolyticum; e.g. Clostridium histolyticum
G11 and/or
ATCCD 21000m4). It provides nutrients required for sufficient growth of the
bacteria and -
optionally in combination with a feed composition according to the invention -
for the effective
production of one or more proteases, such as e.g. collagenase I, collagenase
II, neutral protease
and/or clostripain. In an embodiment, by fermentation of Hathewaya histolytica
(Clostridium
histolyticum) with the inventive culture medium, and optionally with the
inventive feed
composition, the activity of collagenase in the supernatant is 400 PZ units/L
or more, in another
embodiment 600 PZ units/L or more, in another embodiment 1200 PZ units/L or
more. 1 PZ
Unit according to Wuensch catalyzes the hydrolysis of 1 limo! 4-
phenylazobenzyl- oxycarbonyl-
L-prolyl-L-leucyl- glycyl-L-prolyl-D-arginine (PZ) per minute at 25 C, pH 7.1
(Wuensch, E. &
Heidrich, H.G. (1963) Hoppe-Seyler's Z. Physiol. Chem. 333, 149-51).
The use of lower concentrations than 2.5 g/L of yeast extract is in principle
possible, but may
reduce bacterial growth and product yield. The use of concentrations higher
than 100 g/L of
yeast extract is in principle possible, but may not provide much benefit in
terms of bacterial
growth or secretion of proteases. In some embodiments, the concentration of
yeast extract in
the liquid culture medium is in the range of 2.5 to 80, 5.0 to 60 or 10 to 30
g /L. In another
embodiment, the concentration of yeast extract in the liquid culture medium is
in the range of
17 to 25 g /L. In some embodiments, the liquid culture medium comprises 20 g/L
yeast extract.
In principle, any yeast extract can be used. Yeast extracts and methods for
the preparation are
well known in the art. Contrary to animal peptones or plant peptones, yeast
extracts are
produced by better controlled industrial processes which include fermentation
of the yeast in
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an industrial bioreactor. For this reason, batch reliability is higher than
for plant- or animal-
derived components. Different batches of yeast extract produced by the same
process provide
a similar performance in terms of bacterial growth and production of
proteases. Furthermore,
yeast extracts produced by different processes and/or different producers can
substitute each
other and provide similar performance in terms of bacterial growth and
production of
proteases. In summary, yeast extracts provide increased process robustness
over animal
peptones and plant peptones.
Especially, any commercially available yeast extract may be used. Non-limiting
examples are
"BD Yeast Extract Technical" (Becton Dickinson and Company, Miami, FL, USA,
Art.-No.:
288610), "BD Yeast Extract" (Becton Dickinson, Art-No.: 212730), õBD Bacto
Yeast Extract"
(Becton Dickinson, Art.-No.: 212730). "BD Yeast Extract Technical" (Becton
Dickinson, Art.- No.:
288610) provides a high yield of collagenases and other proteases and
fermentations with this
yeast extract are well reproducible. Further suitable yeast extracts are e.g.
Difco Yeast Extract,
low-dusting (LD) Art.-No.: 210933", yeast extracts produced by Ohly, the Kerry
HY-Yeast series,
and the Yeast Extract Art.-No.: A1552 from AppliChem.
If not otherwise mentioned in this text, the yield and concentration of
collagenase is measured
by the PZ-activity of the supernatant. The activity of neutral protease in the
supernatant can
be measured e.g. according to Lin, Y.-C. et al. (1969) J. Biol. Chem. 244, 789-
93; or Kunitz, M.
1947. Crystalline soybean trypsin inhibitor. II. General properties. J. Gen.
Physiol. 30:291-310;
or as described in Example 1. The activity of clostripain in the supernatant
can be measured
e.g. as described in Kzedy, F. J. et al. Biochemistry, 1965, 4, 2303-2308; or
as described in
Example 2.
The inventive liquid culture medium comprises glycine or its pharmaceutically
acceptable salts
in a concentration of 1.0 to 30 g/L. The use of lower concentrations than 1.0
g/L of glycine is in
principle possible, but may reduce bacterial growth and may result in lower PZ-
activity in the
supernatant. Higher concentrations of glycine than 30 g/L may have little
benefit for cell
growth or PZ-activity in the supernatant. In one embodiment, the concentration
of glycine in
the inventive liquid culture medium is in the range of 5 to 8 g/L. In another
embodiment, the
concentration of glycine in the liquid culture medium is in the range of 5 to
7 g/L. In some
embodiments, the liquid culture medium comprises 6.5 g/L glycine.
The inventive liquid culture medium comprises arginine or its pharmaceutically
acceptable
salts in a concentration of 1.0 to 35 g/L. The use of lower concentrations
than 1.0 g/L of a rginine
is in principle possible, but may reduce bacterial growth and may result in
lower PZ- activity in
the supernatant. Higher concentrations of arginine than 35 g/L may have little
benefit for cell
growth or PZ-activity in the supernatant. In one embodiment, the concentration
of arginine in
the inventive liquid culture medium is in the range of 5 to 8 g/L. In another
embodiment, the
concentration of arginine in the inventive liquid culture medium is in the
range of 6 to 7 g/L. In
some embodiments, the liquid culture medium comprises 6.7 g/L arginine.
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The inventive liquid culture medium comprises glutamine or its
pharmaceutically acceptable
salts in a concentration of 0.20 to 0.80 g/L. Glutamine has a positive
influence on bacterial
growth and on the PZ-activity in the supernatant. The use of lower
concentrations than 0.20
g/L of glutamine is in principle possible, but may reduce bacterial growth and
may result in
lower PZ-activity in the supernatant. Higher concentrations of glutamine than
0.80 g/L may
have little benefit for cell growth or PZ-activity in the supernatant. In one
embodiment, the
concentration of glutamine in the inventive liquid culture medium is in the
range of 0.25
to0.40 g/L. In another embodiment, the concentration of glutamine in the
inventive liquid
culture medium is in the range of 0.30 to 0.35 g/L. In some embodiments, the
liquid culture
medium comprises 0.32 g/L glutamine.
The inventive liquid culture medium comprises serine or its pharmaceutically
acceptable salts
in a concentration of 0.20 to 1.0 g/L. Serine has a positive influence on
bacterial growth, but a
negative influence on productivity. Productivity, as defined herein, is the
ratio of PZ-activity to
bacterial growth. Bacterial growth can be determined by known methods, e.g. by
measuring
the turbidity. In some embodiments, bacterial growth is determined by
measuring the optical
density at 600 nm. The use of lower concentrations than 0.20 g/L of serine is
in principle
possible, but may result in lower bacterial growth. Serine concentrations
higher than 1.0 g/L
may reduce productivity. In an embodiment, the concentration of serine in the
inventive liquid
culture medium is in the range of 0.37 to 0.50 g/L. In another embodiment, the
concentration
of serine in the inventive liquid culture medium is in the range of 0.37 to
0.47 g/L. In some
embodiments, the liquid culture medium comprises 0.42 g/L serine.
The inventive liquid culture medium comprises threonine or its
pharmaceutically acceptable
salts in a concentration of 0.3 to 3.0 g/L. The lower concentrations of
threonine within this
range provide higher PZ-activity in the supernatant than the higher
concentrations within this
range. The use of lower concentrations than 0.3 g/L of threonine will result
in lower bacterial
growth. Higher concentrations of threonine than 3.0 g/L may reduce the
productivity. In an
embodiment, the concentration of threonine is not higher than 3.0 g/L. In one
embodiment,
the concentration of threonine in the inventive liquid culture medium is in
the range of 0.5 to
1.50 g/L. In another embodiment, the concentration of threonine in the
inventive liquid culture
medium is in the range of 0.8 to 1.2 g/L. In some embodiments, the liquid
culture medium
comprises 1 g/L threonine.
The liquid culture medium according to the invention comprises 0.4 to 12
mmol/L magnesium
ions (Mg2), e.g. 100 to 3000 mg/L of magnesium sulfate heptahydrate (Mg504 x 7
I120). In an
embodiment the liquid culture medium according to the invention comprises 1.2
to 6.9
mmol/L magnesium ions (Me). In an embodiment, the concentration of magnesium
sulfate
heptahydrate in the liquid culture medium is in the range of 300 to 1700 mg/L.
In another
embodiment the liquid culture medium according to the invention comprises 2.8
to 5.3
mmol/L magnesium ions (Me). In another embodiment, the magnesium sulfate
heptahydrate concentration is in the range of 700 to 1300 mg/L. In some
embodiments, the
liquid culture medium comprises 995 mg/L magnesium sulfate heptahydrate. The
liquid culture
.
.
ccording to the invention comprises 0.34 to 3.4 mmol/L cal,
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to 500 mg/L of calcium chloride dihydrate (CaCl2x 2 H20). In an embodiment,
the liquid culture
medium according to the invention comprises 0.68 to 2.0 mmol/L calcium ions
(Ca2+). In an
embodiment, the concentration of calcium chloride dihydrate in the liquid
culture medium is
in the range of 100 to 300 mg/L. In another embodiment, the liquid culture
medium according
to the invention comprises 1.0 to 2.0 mmol/L calcium ions (Ca2+). In another
embodiment, the
calcium chloride dihydrate concentration is in the range of 150 to 300 mg/L.
In some
embodiments, the liquid culture medium comprises 260 mg/L calcium chloride
dihydrate. In
one embodiment the anions for the magnesium and calcium ions may be selected
from the
group consisting of chloride, sulfate, phosphate and acetate may be used. In
other
embodiments magnesium sulfate may be used for magnesium ions and/or calcium
chloride
may be used for calcium ions. The liquid culture medium according to the
invention comprises
0.00095 to 0.0152 mmol/L selenium, e.g. 0.25 to 4.00 mg/L sodium selenite
pentahydrate
(Na2Se03 x 5 H20). In an embodiment, the liquid culture medium according to
the invention
comprises 0.0027 to 0.0061 mmol/L selenium. In an embodiment, the
concentration of sodium
selenite pentahydrate in the liquid culture medium is in the range of 0.70 to
1.60 mg/L. In
another embodiment, the liquid culture medium according to the invention
comprises 0.0038
to 0.0053 mmol/L selenium. In another embodiment, the sodium selenite
pentahydrate
concentration is in the range of 1.00 to 1.40 mg/L. In some embodiments, the
liquid culture
medium comprises 1.17 mg/L sodium selenite pentahydrate. In some embodiments
sodium
selenite (Na2Se03) or its pentahydrate is used to provide selenium to the
inventive liquid
culture medium, i.e., the medium comprises sodium selenite. Other selenium
comprising
compounds or compositions may be used. It is well within the knowledge of the
expert in the
art to choose suitable forms of selenium. It is well known that the selenium
introduced into
the media may be in a suitable form that has sufficient bioavailability. This
makes the selenium
comprised in the media available to the bacteria. Whether a compound or a
composition
provides sufficient selenium to the media can simply be determined by
comparison of the yield
of the desired proteases in a fermentation. This yield can be compared with
the yield obtained
through use of sodium selenite. If necessary, the amounts of compounds or
composition other
than sodium selenite or its pentahydrate used to provide selenium could be
adjusted so that
the same or similar results are obtained.
The inventive liquid culture medium as described herein may comprise water,
for example,
purified water or water for injection. Purified water is waterthat has been
mechanically filtered
or processed to remove impurities and make it suitable for use, especially
pharmaceutical use.
Water for injection is even more purified and is further defined e.g. in the
European or United
States Pharmacopeia. In one embodiment, the liquid culture medium comprises
water in the
amount of at least 50, 60, or 70 percent by weight based on the whole weight
of the liquid
culture medium. In some embodiments, the liquid culture medium comprises water
in the
amount of at least 78% by weight, Olin the amount of at least 90% by weight,
or in the amount
of at least 95 % by weight. In some embodiments, water is the remainder of the
liquid culture
medium. In some embodiments, the liquid culture medium comprises purified
water or water
for injection according to the European or United States Pharmacopeia, e.g. in
the above
specified amounts.
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In some embodiments, the liquid culture medium is used for the fermentation of
Hathewaya
histolytica (Clostridium histolyticum) and the production of at least one
protease selected from
the group consisting of collagenase I, collagenase II, neutral protease and
clostripain.
In some embodiments, the liquid culture medium comprises, essentially consists
of, or consists
of 10 to 30 g /L of yeast extract, 5 to 8 g/L of glycine, 5 to 8 g/L of
arginine, 0.25 to 0.40 g/L
of glutamine, 0.37 to 0.50 g/L of serine, 0.50 to 1.50 g/L of threonine, 1.2
to 6.9 rnmol/L
magnesium ions (Me), 0.68 to 2.0 mmol/L calcium ions (Ca'), 0.0027 to 0.0061
mmol/L
selenium, and water. In some embodiments, the liquid culture medium comprises,
essentially
consists of, or consists of 10 to 30 g /L of yeast extract, 5 to 8 g/L of
glycine, 5 to 8 g/L of
arginine, 0.25 to 0.40 g/L of glutamine, 0.37 to 0.50 g/L of serine, 0.50 to
1.50 g/L of threonine,
300 to 1700 mg/L of magnesium sulfate heptahydrate (MgSO4 x 7 H20), 100 to 300
mg/L of
calcium chloride dihydrate (CaCl2 x 2 H20), 0.70 to 1.60 mg/L of sodium
selenite pentahydrate
(Na2Se03 x 5 H20), and water. In some embodiments, the liquid culture medium
comprises,
essentially consists of, or consists of 17 to 25 g /L of yeast extract, 5 to 7
g/L of glycine, 6 to 7
g/L of arginine, 0.30 to 0.35 g/L of glutamine, 0.37 to 0.47 g/L of serine,
0.8 to 1.2 g/L of
threonine, 2.8 to 5.3 mmol/L magnesium ions (Mg'), 1.0 to 2.0 mmol/L calcium
ions (Ca2+),
0.0038 to 0.0053 mirnol/L selenium, and water. In some embodiments, the liquid
culture
medium comprises, essentially consists of, or consists of 17 to 25 g /L of
yeast extract, 5 to 7
g/L of glycine, 6 to 7 g/L of arginine, 0.30 to 0.35 g/L of glutamine, 0.37 to
0.47 g/L of serine,
0.8 to 1.2 g/L of threonine, 700 to 1300 mg/L of magnesium sulfate
heptahydrate, 150 to 300
mg/L of calcium chloride di hydrate, 1.00 to 1.40 mg/L of sodium selenite
pentahyd rate, and
water. In some embodiments, the liquid culture medium comprises, essentially
consists of, or
consists of 20 g/L yeast extract, 6.5 g/L glycine, 6.7 g/L arginine, 0.32 g/L
glutamine, 0.42 g/L
serine, 1 g/L threonine, 995 mg/L magnesium sulfate heptahydrate, 250 mg/L
calcium chloride
dihydrate, and 1.17 mg/L sodium selenite penta hydrate, and water (e.g.
purified water).
Since the yeast extracts can be prepared in a reproducible way, the inventive
liquid culture
medium provides a stable and reproducible culture medium with a high batch
stability, which
provides for a high process robustness for the cultivation of Hathewaya
histolytica (Clostridium
histolyticum) and the production of proteases, such as e.g. collagenase I,
collagenase II, neutral
protease and clostripain. In some embodiments, the liquid culture medium is
free from any
components derived from animals or plants. Such animal or plant derived
ingredients may
comprise adventitious agents and/or their ingredients may vary between
manufacturers and
even between different batches of the same manufacturer. Using a liquid
culture medium
without animal or plant derived ingredients for the cultivation of Hathewaya
histolytica
(Clostridium histolyticum) and the production of proteases provides more
safety for the end
user of the proteases and improves reliability of the inventive liquid culture
medium in
fermentations.
The terms "derived from animals" or "animal derived" or "animal -derived" as
used herein shall
mean any substance of animal origin. The terms "derived from plants" or "plant
derived" or
"blant-derived" as used herein shall mean any substance of plant origin. In
some embodiments.
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the terms "derived from animals" or "animal derived" or "animal-derived" as
used herein shall
mean any substance of animal origin and any substance of non-animal origin
processed using
one or more animal-derived substances (e.g. animal-derived enzymes). In some
embodiments,
the terms "derived from plants" or "plant derived" or "plant-derived" as used
herein shall mean
any substance of plant origin and any substance of non-plant origin processed
using one or
more plant-derived substances (e.g. plant-derived enzymes). The terms
"component" or
"ingredient" as used herein shall mean any substance of content of the liquid
culture medium
or the liquid feed composition.
In some embodiments, the liquid culture medium or the liquid feed composition
does not
comprise glucose. In some embodiments, the liquid culture medium or the liquid
feed
composition does not comprise a sugar selected from the group consisting of
glucose,
galactose, lactose, mannose, raffinose, fucose, sucrose and arabinose. In some
embodiments,
the liquid culture medium or the liquid feed composition does not comprise any
sugar.
In a further embodiment, the liquid culture medium comprises an anti-foaming
agent. Anti-
foaming agents suppress the development of foam in the culture medium and
during
fermentation. Any commercially available anti-foaming agent may be used. One
suitable anti-
foaming agent is XIAMETERT" ACP-1500 (EU) Antifoam Compound from Dow. The anti-
foaming
agent may be added in an amount suitable to suppress foaming to an acceptable
level.
Fermentations in small scale may be conducted without the addition of anti-
foaming agent. In
large-scale fermentations anti-foaming agent may be added.
In some embodiments, the liquid culture medium has a pH value in the range of
6.5 to 8.2. In
some embodiments, the pH value of the liquid culture medium is in the range of
7.2 to 8.1 or
7.5 to 8Ø The pH value may be regulated by any known method, e.g. by
addition of a buffer
with a suitable buffer capacity or by addition of acids or bases. In some
embodiments, for
example in pre-cultures and in fermentations with supernatants with a volume
of about 0.5
liter or less, buffer may be added. For example, a 3-(N-
morpholino)propanesulfonic acid buffer
(MOPS buffer) may be used. In some embodiments, for example in a fed-batch
process and/or
in fermentation processes in which the volume of the supernatant is more than
about 1 liter,
the pH value may be continuously measured and acids or bases (or both in the
time course of
a cultivation process) are added to keep the pH value in the desired range.
For example,
phosphoric acid and/or sodium hydroxide may be used. In some embodiments, for
example in
fermentation processes in which the volume of the supernatant is between about
0.5 and
about 1 L, any of the two methods may be used.
In a further embodiment, the liquid culture medium is sterilized. In some
embodiments, the
composition is free from any self-replicating organism. Sterilization can be
achieved by
standard methods known to the skilled person, e.g. by heat treatment, such as,
for example,
autoclaving. In some embodiments, the sterilized liquid culture medium
comprises an
inocul um of Hathewaya histolytica (Clostridium histolyticum). In some
embodiments, the liquid
culture medium is free from any self-replicating organism but Hathewaya
histolytica
histolyticum). In some embodiments, such compositions
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an inoculum of Hathewaya histolytica (Clostridium histolyticum) to a
sterilized liquid culture
medium of the present invention.
In a second aspect, the present invention concerns a liquid feed composition,
for example for
use as feed in a fed-batch process for the cultivation of Hathewaya
histolytica (Clostridium
histolyticum), comprising, essentially consisting of, or consisting of
at least 20 g/L of yeast extract, at
least 3 g/L of threonine,
at least 1.5 g/L of serine, and
water.
The term "fed-batch culture" as used herein shall mean an operational
technique in
biotechnological processes where one or more nutrients are fed (supplied) to a
container,
e.g. a bioreactor, during cultivation and in which the product(s) remain in
the container until
the end of the run. For further details on fed-batch processes see e.g. Tsuneo
Yamane and
Shoichi Shimizu (Fed-batch Techniques in Microbial Processes; Advances in
Biochem
Eng./Biotechnol 1984, 30:147-194).
The liquid feed composition and the liquid culture medium are different
compositions. In some
embodiments, the liquid feed composition comprises at least 30, 40, or 50 g/L
yeast extract. In
some embodiments, the liquid feed composition comprises at least 4, 5, 6, 7,
8, 9, or 10 g/L
threonine. In some embodiments, the liquid feed composition comprises at least
2.0, 2.5, 3.0,
4.5, 5.0 g/L serine. In some embodiments, the liquid feed composition
comprises, essentially
consists of, or consists of 50 to 200 g/L yeast extract, 3 bis 60 g/L
threonine and 1.5 to 40 g/L
serine, and water. In some embodiments, the liquid feed composition comprises,
essentially
consists of, or consists of 70 to 130 g/L yeast extract, 3 bis 10 g/L
threonine and 1.5 to 10 g/L
serine, and water. In some embodiments, the liquid feed composition comprises,
essentially
consists of, or consists of 100 g/L yeast extract, 5 g/L threonine and 2.5 g/L
serine, and water.
The inventive liquid feed composition as described herein comprises water, for
example
purified water or water for injection, in the amount of at least 50, 60, or 65
percent by weight
based on the whole weight of the liquid feed composition. In some embodiments,
the liquid
feed composition comprises water, for example purified water or water for
injection, in the
amount of at least 70 percent by weight, or at least 80 percent by weight, or
at least 95 % by
weight. In some embodiments, water is the remainder of the liquid feed
composition.
In some embodiments, the liquid feed composition has a pH value in the range
of 6.5 to 8.2.
In a further embodiment, the liquid feed composition is sterilized. In some
embodiments, the
composition is free from any self-replicating organism. Sterilization can be
achieved by
standard methods known to the skilled person, e.g., by heat treatment, such
as, for example,
autoclaving.
In a third aspect, the present invention concerns a kit of parts comprising a
liquid culture
medium of the present invention and a liquid feed composition of the present
invention. The
is useful, for example, for the fermentation of Hathewayc
I r."-1-'; '
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histolyticum; e.g. Clostridium histolyticum G11 and/or ATCC 21000TM) and the
production of
one or more proteases, e.g. in a fed-batch process.
A fourth aspect of the present invention is a supernatant of a Hathewaya
histolytic
(Clostridium histolyticum, e.g. Clostridium histolyticum G11 and/or ATCC
210001M) liquid
culture, comprising a liquid culture medium of the present invention and/or a
liquid feed
composition of the present invention and one or more proteases. The one or
more proteases
may be selected from the group consisting of collagenase I, collagenase II,
neutral protease
and clostripain. In some embodiments, the supernatant of a Hathewaya
histolytica
(Clostridium histolyticum) liquid culture prepared by use of a liquid culture
medium and/or a
liquid feed composition of the present invention has a collagenase activity of
at least 400 PZ
Units/L (Wuensch units), or at least 500 PZ Units/L, or at least 600 PZ
Units/L, or at least 700
PZ Units/L, or at least 800 PZ Units/L, or at least 900 PZ Units/L, or at
least 1000 PZ Units/L, or
at least 11000 PZ Units/L, or at least 1200 PZ Units/L.
An embodiment of this aspect of the invention is a culture supernatant of
Hathewaya
histolytica (Clostridium histolyticum) comprising one or more proteases
obtainable by the
method of (a) providing a sterilized liquid culture medium according to the
invention with an
inoculum of Hathewaya histolytica (Clostridium histolyticum), (b) cultivating
the bacteria,
whereby the bacteria secrete the one or more proteases into the liquid phase;
(c) separating
solids, e.g. cellular and other particulate matter, from the liquid phase; and
thereby obtaining
the culture supernatant from Hathewaya histolytic (Clostridium histolyticum)
comprising one
or more proteases. In some embodiments, the supernatant is obtained from a
liquid fed- batch
culture. Accordingly, in some embodiments, the supernatant is obtainable by
the method of
(a) providing a sterilized liquid culture medium according to the invention
with an inoculum of
Hathewaya histolytica (Clostridium histolyticum), (b) cultivating the bacteria
in a fed-batch
operation, (c) adding the liquid feed composition of the invention, (d)
separating solids, e.g.
cellular and other particulate matter, from the liquid phase; and thereby
obtaining the culture
supernatant from Clostridium histolyticum comprising one or more proteases. In
some
embodiments, the one or more proteases may be selected from the group
comprising
collagenase I, collagenase II, neutral protease, and clostripain.
In a fifth aspect, the present invention concerns the use of a liquid culture
medium of the
present invention for cultivating Hathewaya histolytica (Clostridium
histolyticum; e.g.
Clostridium histolyticum G11 and/or ATCC zlooQTM) and obtaining at least one
protease from
the culture supernatant, e.g. a protease with collagenase activity, such as
collagenase I and/or
collagenase II, and/or neutral protease and/or clostripain. In a sixth aspect,
the present
invention concerns the use of a liquid feed composition of the present
invention for cultivating
Hathewaya histolytica (Clostridium histolyticum; e.g. Clostridium histolyticum
G11 and/or
ATCC 21000TM) and obtaining at least one protease from the culture
supernatant, e.g. a
protease with collagenase activity, such as collagenase I and/or collagenase
II, and/or neutral
protease and/or clostripain.
'- - --- ---th aspect, the present invention concerns a method compris'-- +1--
-+-- -F
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- cultivating Hathewaya histolytica (Clostridium histolyticum; e.g.
Clostridium
histolyticum G11 and/or ATCC 21000TM) in a liquid culture medium of the
present
invention and obtaining at least one protease from the culture supernatant.
In some embodiments, collagenase I, collagenase II, neutral protease and/or
clostripain are
obtained with the aforementioned method. In some embodiments of the inventive
method, the
pH value of the culture is controlled such that it is in the range of 6.5 to
8.2. In some
embodiments, the pH value of the culture is controlled such that it is in the
range of 7.2 to 8.1
or 7.5 to 8.
In some embodiments of the method of the present invention, the cultivation is
preferably
performed in a fed-batch operation by use of a sterilized liquid feed
composition as described
herein as feed. In some embodiments of such a fed-batch process, the pH value
of the culture
is controlled such that it is in the range of 7.2 to 7.8.
In some embodiments, the method according to the present invention comprises
the steps of
(a) providing a sterilized liquid culture medium according to the present
invention with
an inoculum of Hathewaya histolytica (Clostridium histolyticum bacteria; e.g.
Clostridium histolyticum G11 and/or ATCC 21000T");
(b) cultivating the bacteria, optionally adding a liquid feed composition
according to
the present invention, whereby the bacteria secrete the one or more proteases
into the
liquid phase;
(c) separating solids, e.g. cellular and other particulate matter, from the
liquid phase,
thereby obtaining a supernatant;
(d) obtaining the one or more proteases from the supernatant;
thereby producing the one or more proteases from Hathewaya histolytica
(Clostridium
histolyticum).
The culture or fermentation process may be monitored continuously or at one or
more time
points during cultivation, e.g. by determining turbidity and/or by determining
protease activity
in a sample of the culture supernatant (which may be taken at one or more time
points during
cultivation). In some embodiments, the addition of the liquid feed composition
is started when
bacterial growth rate has reached the exponential phase. In some embodiments,
the addition
of the liquid feed composition is started when bacterial growth
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rate has reached about 40%, 50%, 60%, or 70% of the turbidity maximum. The
bacteria may be
cultivated for 15 to 19 hours. Since the enzyme ratio changes over time, the
cultivation time
may be adapted according to the one or more target enzymes. Suitable methods
to monitor
the cultivation and bacterial growth phases are e.g. disclosed in EP 2 133 415
Al. The one or
more proteases may be obtained from the culture supernatant by any known
methods, e.g. in
crude form or purified. Accordingly, in some embodiments, step (d) may
comprise one or more
purification steps such as e.g. filtration and/or column chromatography. WO
2020/164721 Al
describes some example procedures for obtaining one or more proteases from a
culture
supernatant of Hathewaya histolytica (Clostridium histolyticum).
Examples:
Analytic methods:
Determination of Wuensch activity in the supernatant has been conducted
according to
Wuensch, E., Heidrich, H.G.; Hoppe-Seyler's Zeit. Physiol. Chem., 333, 149-51
(1963). 1 PZ unit
activity according to Wuensch is the activity that catalyzes the hydrolysis of
1 p.mol 4- phenyl-
azobenzyloxycarbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-arginine per minute
at 25 C, pH 7.1.
All values of Wuensch units herein are given in PZ Units/liter (U/L), i.e.
Wuensch units per liter
of culture medium, if not otherwise stated.
The activities of clostripain and neutral protease have been determined as
described in
Examples land 2.
The turbidity was measured off-line in the Mettler Toledo UV-VIS
spectrophotometer
UV5Nano at 600 nm (OD 600). It is measured against an empty sample (medium
without
inoculum). If necessary, the solution must be diluted so that the measured
value is in the
measuring range of up to 0.6 OD 600.
Materials:
In all experiments Clostridium histolyticum Gil and/or ATCC 21000TM were
used. ATCC
21000" is available at the ATCC (American Type Culture Collection). As yeast
extract "BD Yeast
Extract" (Becton Dickinson, Art.-No.: 288610) was used. In the below described
experiments
all cultures were inoculated with 2 volume percent of a pre-culture. The pre-
culture was
prepared by Inoculation of the liquid culture medium with the contents of 1
vial (the vials were
defined to contain a minimum of 106 CFU; according to analysis the vials
contained 35 x 106
CFU/Vial.) of lyophilized Hathewaya histolytica (Clostridium histolyticum G11
or ATCC
210001M) mixed with 1 mL of the liquid culture medium and anaerobic incubation
at 37 C in
shake flasks.
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As bioreactors Multifors bioreactors (from Infors GmbH, Einsbach, Germany)
were used. Further
materials, reagents and devices are described in the Examples below.
Example 1: Activity assay of neutral protease using a continuous photometric
assay with
the substrateN-(34furyl] acryloyI)-glycine leucine amide (FAGLA).
Principle: The neutral protease from Hathewaya histolytica (or Clostridium
histolyticum)
catalyzes the hydrolysis of the peptide bond inthe substrate FAGLA between the
amino acids
glycine and leucine. In the photometric assay, substrate conversion was
measured
continuously by decreasing absorbance at 345 nm. The decrease in absorbance or
the
resulting negative slope is thus a direct measure of substrate conversion and
thus of enzyme
activity.
Units definition: One FAGLA unit (1U) is defined as the hydrolysis of 1p.mol
of N-[3-(2-
furypacryloy1]-glycine-L- leucine amide per minute.
Devices: temperature controlled UV-V1S spectrophotometer (e.g. Cary 50, 60 or
100, Agilent),
circular shaker (e.g. Scientific Industries, Vortex Genie2), UV cuvettes semi-
micro (e.g. Brand,
Art.No. 759150), semi-micro quartz cuvettes (e.g. Hellma), PD-10 Desalting
Columns (e.g. GE
Healthcare, Art. No. 17-0851-01).
Reagents: purified water, Ph. Eur., 2-Morpholinoethanesulfonic acid
monohydrate, abbrev.
MES (e.g. Merck, Art. No. 7 1.060126), Calcium chloride dihydrate (e.g. Merck,
Art. No.
1.02382), Dimethyl sulfoxide for spectroscopy, abbreviation DIMS (e.g. Merck,
Art. No.
1.02950.), Triton X-100 (e.g. Sigma-Aldrich, Art. No. 23,472-9, 2-propanol
(e.g. Merck, Art. No.
1.01040), 1 mol/1 Sodium hydroxide solution (Merck, Art. No. 1.09137), FA-Gly-
Leu-NH2 (e.g.
Bachem, Art. No. 4003615), current reference substance Neutral Protease NB
(REF00236),
Tris(hydroxymethyl)aminomethane, abbreviation Iris (e.g. Merck, Art. No.
1.08382.), 1 mol/1
hydrochloric acid (e.g. Merck, Art. No. 1.09057).
Reagent solutions:
a) MES buffer pH 6.5: 10.67 g (0.05 mol) of 2-morpholinoethanesulfonic acid
monohyd rate
and 0.735 g (0.005 mol) of calcium chloride dihydrate were dissolved in
approximately 800 ml
of purified water. Then 10 ml of 2- propanol (1% (v/v)) was added to the
buffer mixture. The
pH was adjusted to 6,5 at room temperature with 1 mol/1 sodium hydroxide
solution and filled
up to 1000 ml with purified water. 0.1 ml of Triton X-100 were added to the
buffer while
stirring by using a 100 p.1 piston-stroke pipette to slowly pipette the highly
viscous Triton X-
100 solution (0.01% (v/v). The detergent was added to the buffer mixture and
at the end the
tip is dropped into the buffer. The solution was stirred until homogeneity was
reached. The
obtained buffer solution has a concentration of 0.05 mol/12-
morpholinoethanesulfonic acid
and 0.005 mol/1 of calcium chloride.
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b) FAG LA substrate solution (concentration = 2.5 mmo1/1): First, a 0.1M FAGLA
solution was
prepared (36.9 mg FAGLA dissolved in 1.2 mldimethyl sulfoxide). Then 1 ml of
0.1 M FAGLA
solution was mixed with 39 ml of MES buffer pH 6.5, as described under a).
c) Reference substance: Approximately 12-20 mg of the reference substance
Neutral
Protease NB (Nordmark Biochemicals, Uetersen, Germany) was weighed and
adjusted to a
concentration of 10 mg/ml with MES buffer pH 6.5 as described under a). This
solution was
stored on ice. Further dilutions (within the valid working range, as described
below) were
prepared from this solution with MES buffer pH 6.5. Approximately 2.5 to 3.6
mg/ml of
reference substance was used. The absorbance changes must lie within the valid
working
range. The analyte solutions were prepared shortly before the determination
with MES buffer
p1-16.5 at RI and measured promptly. The activity of the reference substance
was measured
in duplicate for at least one dilution in the valid working range.
d) Iris buffer pH 9.5: 0.606 (0.005 mol) tris(hydroxymethyl)aminomethane and
0.37 g (0.0025
mol) calcium chloride dihydrate was dissolved in approximately 400 ml purified
water. The pH
was adjusted to 9.5 with 1 mol/1 hydrochloric acid at room temperature and
filled up to 500
ml with purified water. The resulting buffer had a concentration of
tris(hydroxymethyl)aminomethane of 0.01 mol/land of Calcium chloride of 0.005
mol/l).
Sample preparation:
At least one duplicate determination was carried out. Depending on the
activity, the samples
were diluted to be within the valid working range (see below). Samples from
the concentrate
of a cell-free culture supernatant were re-buffered with a PD- 10 column
equilibrated with
Tris buffer pH 9.5 before measurement.
Implementation:
The test was performed at +30 C and a wavelength of 345 nm in a temperature-
controlled
UV-VIS spectrophotometer. The absorbance change at 345 nm was determined for a
time
interval of 5 min.
The FAGLA substrate solution was tempered for approx. 30 min in a water bath
at +30 C
before the actual measurement. Alternatively, the semi-micro cuvettes incl.
0.8 ml substrate
solution can be tempered directly in the UV-VIS photometer for at least 10 min
before
measurement. Before starting a measuring series, the photometer was calibrated
against MES
buffer pH 6.5 without substrate solution. For the measurement, 0.8 ml of
tempered substrate
solution was placed in the cuvettes. Then 0.2 ml sample solution was added,
immediately
premixed by drawing up with the pipette (approx. three piston strokes) and the
entire reaction
mixture was mixed with a disposable stirring spatula. Then the measurement was
started
immediately. The final volume in the test was 1 ml. At least one duplicate
determination was
carried out on each sample. The specified substrate concentration of 2 mmo1/1
was below the
saturation value, i.e. the measuredspecific activity depends on the substrate
concentration.
Therefore, the substrate concentration in the assay must be strictly adhered
to and only
activities determined at identical substrate concentrations are comparable.
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16
Valid Working Range:
A prerequisite for an evaluable activity measurement is an absorbance change
per minute (A
A 345nm / min) in the valid working range from - 0.01 to - 0.035 and the
course of the negative
slope must be linear. If the values are too high (A A 345nm / min), stronger
dilutions must be
used and if the values are too low, lower dilutions are to be used.
Evaluation:
The current reference substance was evaluated. First, a target value and
acceptance criteria
were defined. The activities of the reference substance and the samples from
the mean values
of the duplicate determination were calculated. To calculate the negative
absorbance change
(AA/ min) of the measurement, values from 0 to 5 min were evaluated.
Acceptance criteria
for evaluation: A A 345nm / min of all measured values must lie in the linear
working range.
The maximum deviation may be 5 8% of the mean value. If there were major
deviations, the
measurements were repeated.
The following formula was used to calculate the activity of samples with
unknown
concentration:
A A = V - VF
Neutral Protease Activity = __________
E V
The following formula was used to calculate the activity of samples with known
weights:
AA-V-VF
Neutrat Protease Activity [1.1/Ing
E = V = In
A A =Absorption change per minute at 345 nm VF
= Dilution factor of the sample
V = 1 ml (total volume)
v = 0.2 ml (sample volume)
= -0.317 mM-1 cm-1 absorption coefficient of FAGLA m
= sample weight in mg
Example 2: Determination of clostripain activity in culture supernatant from
Clostridium
histolyticum
Principle:
The enzyme clostripain catalyzes the hydrolysis of benzoyl-L-arginine ethyl
ester (BAEE) in the
presence of calcium and the reducing agent dithiothreitol (DTT), as measured
photometrically
at 255 nm (Kezdy FJ, Lorand L, Miller KD. Titration of active centers in
thrombin solutions.
Standardization of the enzyme. Biochemistry 1965;4:2302-8). The increase in
absorbance per
unit time is a direct measure of enzyme concentration when the reaction is of
pseudo-zero
order, that is, when it is constant. One unit is the amount of enzymatic
activity that
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catalyzes the hydrolysis of 1 p.mol BAEE per minute at 25 C and pH 7.8 in the
presence of
2.5 mM DTT.
Devices: Spectrophotometer Cary 50 (Varian) with temperature-controlled
cuvette block
Vortexer, Rotator, Quartz cuvettes, d = 10 mm.
Reagents: Purified water, Ph. Eur. and USP, Dithiothreitol (DTT) (Serva, Art.
No. 20710), Na-
Benzoyl-L-arginine-ethyl ester HCI (BAEE) (Serva, Art. No. 14600), Sodium
dihydrogen
phosphate monohydrate (Merck, Art. No. 6346), 1 N Sodium hydroxide solution
(Merck, Art.
No. 09137), Calcium acetate hydrate (Kraft, Art. No. 15208)
Reagent solutions: 7.5 mM DTT solution: 57.8 mg DTT were dissolved to 50 ml
purified water.
The solution was prepared fresh daily. 1.5 mM BAEE solution: 51.8 mg BAEE were
dissolved
to 100 ml purified water. The solution was prepared fresh daily. 75 mM sodium
phosphate
buffer, pH 7.8: 10.35 g of sodium dihydrogen phosphate was dissolved in
approximately 800
ml of purified water and the pH was adjusted accurately to pH 7,8 with sodium
hydroxide
solution at room temperature. The solution was then filled up to 1000 ml with
purified water.
Enzyme solvents: Solution A (without activation): 1.0 mM Ca(0Ac)2: 40 mg
calcium acetate
hydrate were dissolved to 250 ml purified water. Solution B (with activation):
1.0 mM Ca(0Ac)2
with 5 mM DTT: 38.4 mg DTT was dissolved with solution A to 50 ml. Solution C
(with
activation): 1.0 mM Ca(0Ac)2 with 2.5 mM DTT: 19.2 mg DTT was dissolved with
solution A to
50 ml. The solutions were prepared fresh daily.
Reference substance: For each analysis, a Collagenase NB1 (Nordmark
Biochemicals,
Uetersen, Germany) as reference substance for identity was also analyzed.
Sample Preparation: The protein concentration in the cell-free culture
supernatant was
determined by measuring absorbance at 280 nm by spectrophotometry. Three
samples of the
cell-free culture supernatant where taken and each sample was diluted with
Solution B to a
concentration of 20 mg of the sample per ml of Solution B.
Each of those diluted samples was split as follows:
500 l sample + 500 pi Solution B (Sample X) 500
per sample I sample + 500 p.I Solution A (Sample Y)
weight
Sample X and Sample Y are incubated between 40 minutes and 4 hours at +4 C
and then
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100 ill of Sample X are further diluted with 900 pi of Solution C and 200
1..t1 of Sample Y are
further diluted with 200111 of Solution A to obtain Samples X and Y as ready
for measurement
("Sample Xm" and "Sample Ym").
Implementation:
The activated Sample Xm and the non-activated Sample Ym were measured in
parallel. In
addition a zero measurement was performed. The following amounts of reagents
(pre-
tempered to 25 C) were pipetted into quartz cuvettes:
Reagent Zero for for
measure Sample Sampl
ment Ym (not e Xm
activated (activ
ated)
NaH2PO4 buffer, pH 1.0 1.0 1.0
7.8 ml ml ml
7.5 nn M DTT 1.0
ml
1.5 mM BAEE 1.0 1.0 1.0
ml ml ml
Purified water 1.1 1.0
ml ml
Sample Xm 0.1
ml
Sample Ym 0.1
ml
Sample Xm and Sample Ym, respectively, were added last, the content of the
cuvettes was
mixed and the change in absorbance was measured immediately after mixing at
255 nm for 5
min at 25 C. The slope AA 255 nm/min was evaluated in the linear region of the
curve. The
measured slopes of the absorbance at 255 nm per minute of the samples must be
linear over the entire 5 minutes, otherwise dilutions must be adapted.
Calculation of the activity:
The following formula was used to calculate the activity:
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AA * V,* dilution factor
Clostripain activity [u / m/]=
0.81* Vp
AA25.5 nm slope per minute at 255 nm
VG - total volume used in the test (VG = 3.1 ml)
Vp - volume of sample used in the test (Vp = 0.1 ml)
0.81 - molar absorption difference of BAEE against benzoyl-L-
arginine at 255 nm, i.e.
810 M-lcm-1
dilution factor: factor of all dilutions beginning with the samples
of the cell-free culture
supernatant until the measurement
Example 3: Preparation of a liquid culture medium
20 g A of yeast extract, 6.5 g/L of glycine, 6.7 g/L of a rginine, 0.32 g/L of
glutamine, 0.42 g/L of
serine, 1.00 g/L of threonine, 995 mg/L of magnesium sulfate hepta hydrate
(MgSO4 x7 H20),
260 mg/L of calcium chloride dihydrate (CaCl2 x 2 H20) and 1.17 mg/L of sodium
selenite
pentahydrate (Na2Se03 x 5 H20) and 10 mg of a silicone-based anti-foaming
agent (XIAMETERT"
ACP-1500 (EU) Antifoam Compound) were added to a 1 liter flask pre-filled with
about 600 ml
purified water and the flask was subsequently filled with purified water under
stirring up to an
overall volume of the liquid culture medium of one liter. Finally the liquid
culture medium was
sterilized in an autoclave. After sterilization, the pH was adjusted to 7.5 by
addition of a 10 M
sodium hydroxide solution in water.
Example 4: Preparation of a liquid culture medium for pre-cultures
20 g of yeast extract, 6.5 g of glycine, 6.7 g of a rginine, 0.32 g of
glutamine, 0.42 g of serine,
1.00 g of threonine, 995 mg of magnesium sulfate heptahydrate (Mg504 x 7 H2O),
260 mg of
calcium chloride dihydrate (CaCl2 x2 H20), 1.17 mg of sodium selenite
pentahydrate (Na2Se03
x 5 H20) and 41.86 g 3-(N-Morpholino)propanesulfonic acid buffer and 10 mg of
a silicone-
based anti-foaming agent (XIAMETERTm ACP-1500 (EU) Antifoam Compound) were
added to a
1 liter flask pre-filled with about 600 ml purified water. The flask was
subsequently filled with
purified water under stirring up to an overall volume of the liquid culture
medium of one liter
and the pH was adjusted to 7.9 by addition of a 10 M sodium hydroxide solution
in water.
Finally the liquid culture medium was sterilized in an autoclave.
Example 5: Preparation of a liquid feed composition
100 g of yeast extract, 5 g of threonine and 2.5 g of serine were added to a 1
liter flask pre-
filled with about 600 ml purified water and the flask was subsequently filled
with purified water
under stirring up to an overall volume of the liquid culture medium of one
liter. Finally the
-..ifure medium was sterilized in an autoclave.
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Example 6: First pre-culture
A 100 mL Erlenmeyer flask was filled with 100 mL medium according to Example
4. About 1 mL
of the medium was removed from the flask and the contents of one vial of
lyophilized
Clostridium histolyticum G11 (about 35 x 106 CFU/Vial) or ATCC 21000TM was
mixed therewith.
The medium was inoculated with this mixture. The culture thus prepared was
grown without
shaking for 24 hours ( 1 hour). Growth took place either in an anaerobic pot
with exclusion of
oxygen (Anaerocult, manufacturer Merck) in an incubator or in an anaerobic
workbench with
anaerobic gas atmosphere (nitrogen 85%, carbon dioxide 10% and hydrogen 5%).
The ambient
temperature was kept at 370C.
Example 7: Second pre-culture
For the fermentation in the 1 L bioreactor, a 100 mL Erlenmeyer flask was
filled with 100 mL
medium according to Example 4. The medium was inoculated with 5 mL of the
first pre-culture
(which corresponds to 5% of the second pre-culture medium volume). The culture
was grown
without shaking for 12 hours ( 0.5 hours). Growth took place under anaerobic
conditions as
disclosed in Example 6 and at an ambient temperature of 37 C. For the
fermentation in the 30
L bioreactor, the second pre-culture was prepared in a 1000 mL flask filled
with 1000 mL
medium according to Example 4. The medium was inoculated with 50 mL of the
first pre-
culture (which corresponds to 5% of the second pre-culture medium volume). The
culture was
grown without shaking for 12 hours ( 0.5 hours). Growth took place under
anaerobic
conditions as disclosed in Example 6 and at an ambient temperature of 37 C.
Example 8: Main culture in a bioreactor
For the fermentation in the 1 L bioreactor, the start volume of the medium
(0,6 L) was
inoculated with 12 mL of the second pre-culture (which corresponds to 2% of
the medium
volume). For the fermentation in the 30 L bioreactor, the start volume of the
medium (27 L)
was inoculated with 540 mL of the second pre-culture (which corresponds to 2%
of the medium
volume).
The fermentation parameters for the respective system and the respective scale
are shown in
the table below:
Table 1: Parameter for fermentation in two different bioreactors
Bio reactor
Parameter unit Multifors (1 11 Techfors (30
1)
Volume of the L 0,6 27
liquid culture
medium (at the
beginning)
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Volume of mL 12 540
inoculation
with second
pre-culture
Feed rate for g/h 25 916
the liquid
feed
corn position
Nitrogen L/mi 0,05 1,83
atmosphere
vvm 0,083 0,083
Stirrer rpm 330 165
pH value 7,5 7,5
Acids and 10 % H3PO4, 10 % H3PO4,
bases 5mM NaOH 5mM NaOH
used for pH-
control
Temperature C 37 37
The liquid culture medium was introduced into the bioreactor and sterilized
(the Multifors in
an autoclave; the Techfors in the reactor itself). Subsequently the bioreactor
was flushed with
nitrogen and stirred for at least 3 h before inoculation. 2 volume percent of
inoculum of were
used. The corresponding amount of the second pre-culture of Example 7 was
removed from
the second pre-culture using a sterile syringe and the main culture was
inoculated via the
inoculation port of the bioreactor. On a 30 L scale, the pre-culture was
pumped into the
bioreactor via a hose (the container with the pre-culture was placed on a
scale in order to
determine the correct amount of inoculum). The culture time was between 15 and
19 hours,
mainly 17 hours and addition of the liquid feed composition according to
Example 5 was
started after 9 hours of fermentation.
The following table shows the amounts of proteases determined in the
supernatant with
Clostridium histolyticum G11:
1L 30L
Collagenase activity [U/mI1 1,140 1,095
Clostripain activity [U/mL] 4,4 5,4
Neutral protease activity 0,037 0,044
[U/m L]
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It can clearly be seen that the scale up for this process was successful and
can be achieved
without significant loss of activity for any of collagenase, clostripain and
neutral protease.
The following table shows the amounts of proteases determined in the
supernatant with
Clostridium histolyticum ATCC 21000TM:
Neut
CoIla Clost
ral
gena ripai
Culture supernatant Prote
se n
ase
U/m1 Wm!
Wm!
First pre-culture 0,463 - -
Second pre-culture 0,403
Main culture in 30 L
0,310
bio reactor 13h
Main culture in 30 L
0,354 22,8 0,119
bio reactor 15h
Main culture in 30 L
0,425 26,8 0,125
bioreactor 17h
Main culture in 30 L
0,443 30,7 0,146
bioreactor 19h
It can clearly be seen that the large-scale production of enzymes was
successful and can be
achieved with different strains of Clostridium histolyticum.
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